Curcumol enhances cisplatin sensitivity of gastric cancer: involvement of microRNA-7 and the nuclear factor-kappa B/snail family transcriptional repressor 1 axis

Abstract

Cisplatin is a primary chemotherapeutic drug for gastric cancer (GC) patients, but the drug resistance remains the leading cause of treatment failure and high mortality. Curcumol is a bioactive sesquiterpenoid that has reportedly been linked to cisplatin sensitivity in GC. This study focuses on the exact functions of curcumol in the cisplatin sensitivity of GC cells and the molecules of action. The curcumol treatment reduced the viability and migration and enhanced cisplatin sensitivity of GC cells in a dose-dependent manner. Microarray analysis suggested that microRNA-7 (miR-7) was the most upregulated miRNA in GC cells after curcumol treatment. The Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that the curcumol-affected genes, including the target genes of miR-7, were enriched in the nuclear factor-kappa B (NF-κB) pathway, whose activity was suppressed after curcumol treatment. miR-7 was found to target and suppress RELA proto-oncogene (RELA, also known as p65), a NF-κB subunit. Downregulation of miR-7 blocked the sensitizing effects of curcumol on cells to cisplatin and led to increased expression of NF-κB p65 and snail family transcriptional repressor 1 (SNAIL). Further downregulation of RELA enhanced, whereas upregulation of SNAIL suppressed the sensitivity again. In summary, this study suggests that curcumol sensitizes GC cells to cisplatin via miR-7 and the suppression of the NF-κB/SNAIL axis. The findings may offer new thoughts that curcumol in combination with cisplatin might be a useful strategy for GC management.

Keywords: Curcumol; NF-κB/SNAIL; RELA; cisplatin; gastric cancer; miR-7.

Graphical abstract

Figure 1.

Curcumol suppresses growth and migration of GC cells and enhances cisplatin sensitivity. a, proliferation activity of GC cell lines MKN45, HGC27, AGS and NCIN87 and normal gastric cells after different doses of curcumol treatment evaluated by CCK-8 method (*p < 0.05, **p < 0.01; two-way ANOVA); b, migration ability of MKN45 and HGC27 cells after curcumol treatment examined by Transwell assay (*p < 0.05, **p < 0.01; two-way ANOVA); c, proliferation activity of MKN45 and HGC27 cells after co-treatment of curcumol and cisplatin evaluated by CCK-8 method (*#p < 0.05, **##p < 0.01; two-way ANOVA; * vs. 1% alcohol; * vs. the previous dose of curcumol); d, migration of MKN45 and HGC27 cells after co-treatment of curcumol and cisplatin examined by Transwell assay (*#p < 0.05, **##p < 0.01; two-way ANOVA; * vs. 1% alcohol; * vs. the previous dose of curcumol).

Figure 2.

Curcumol upregulates miR-7 expression in GC cells. a, miRNAs with differential expression in cells after curcumol treatment identified by microarray analysis; b, miR-7 expression in cells after 80 μM curcumol treatment examined by RT-qPCR (*p < 0.05; two-way ANOVA); c, miR-7 expression in tumor and the paired normal tissues evaluated by RT-qPCR (n = 33; *p < 0.05; the unpaired t test); d, correlation between miR-7 expression and the patient’s survival (*p < 0.05; Kaplan-Meier analysis); e, miR-7 expression in MKN45 and HGC27 cells after different doses of curcumol treatment examined by RT-qPCR (*p < 0.05, **p < 0.01; two-way ANOVA).

Figure 3.

miR-7 inhibition blocks the function of curcumol in cells. a, transfection of miR-7 inhibitor in cells determined by RT-qPCR (*p < 0.05; two-way ANOVA); b, proliferation of cells evaluated by the CCK-8 method (*p < 0.05; two-way ANOVA); c, migration of cells evaluated by the Transwell assay (*p < 0.05; two-way ANOVA); d, apoptosis of cells detected by the flow cytometry (*p < 0.05; two-way ANOVA); e, E-cadherin and Vimentin protein levels in cells detected by western blot assays (*p < 0.05; two-way ANOVA).

Figure 4.

miR-7 inhibition blocks the function of curcumol in cisplatin sensitization in vivo. a, volume change of the xenograft tumors in nude mice (*p < 0.05; two-way ANOVA); b, tumor weight on the 28^th d (*p < 0.05; two-way ANOVA); c, miR-7 concentration in the collected tumor tissues determined by the FISH assay.

Figure 5.

miR-7 targets RELA to mediate the NF-κB/SNAIL signaling pathway. a, the signaling pathway enriched by the target genes of miR-7 analyzed by the KEGG enrichment analysis; b, expression of the target mRNAs of miR-7 in MKN45 cells after miR-7 inhibition examined by RT-qPCR (*p < 0.05; two-way ANOVA); c, binding between miR-7 and RELA confirmed by luciferase assay (*p < 0.05; two-way ANOVA); d, mRNA expression of RELA in tumor and the adjacent tissues determined by RT-qPCR (n = 33) (*p < 0.05; the paired t test); e, correlation between miR-7 and RELA expression (*p < 0.05; Pearson’s correlation analysis); f, mRNA expression of RELA in MKN45 and HGC27 cells after curcumol treatment detected by RT-qPCR (*p < 0.05, **p < 0.01; two-way ANOVA); g, protein levels of NF-κB p65 and SNAIL in cells after curcumol and miR-7 inhibitor treatments examined by western blot analysis (*#p < 0.05; two-way ANOVA; * vs. 1% alcohol; * vs. the previous dose of curcumol).

Figure 6.

RELA and the NF-κB/SNAIL axis mediates the sensitivity of GC cells to cisplatin in vitro. a, protein levels of NF-κB p65 and SNAIL in cells detected by western blot assays (*#p < 0.05; two-way ANOVA); b, proliferation activity of cells evaluated by the CCK-8 method (*#p < 0.05; two-way ANOVA); c, migration of cells evaluated by the Transwell assay (*#p < 0.05; two-way ANOVA); d, apoptosis of cells examined by the flow cytometry (*#p < 0.05; two-way ANOVA); e, protein level of E-cadherin and Vimentin in cells detected by western blot analysis (*#p < 0.05; two-way ANOVA).

Figure 7.

RELA and the NF-κB/SNAIL axis mediates the sensitivity of GC cells to cisplatin in vivo. a, volume change of the xenograft tumors in mice (*#p < 0.05; two-way ANOVA); b, tumor weight on the 28^th d (*#p < 0.05; two-way ANOVA); c, positive NF-κB p65 and SNAIL expression in the tumor tissues determined by the immunohistochemical staining.